Mechanism of action of clostridial neurotoxins and rational inhibitor design

Abstract

Botulinum neurotoxin type B (BoNT/B) belongs tothe family of botulinum neurotoxins, which containsseven proteins (serotypes A to G). They act on theperipheral nervous system causing botulism, a fre-quently fatal disease (Shone, 1986). These neurotoxinsare proteins of 150 kDa composed of two subunits: a50 kDa light chain responsible for the protein toxicityby blocking the calcium-mediated release of excitatoryneurotransmitters (Dolly et al., 1990), and a 100 kDaheavy chain involved in the binding to the nerveendings, the internalisation and the translocation of thelight chain into the cytosol (Montecucco et al., 1994).The light subunits are zinc-endopeptidases (Schiavo etal., 1992) and cleave specifically one of the three neu-ronal proteins associated with exocytosis: synaptobre-vin, SNAP-25 or syntaxin. BoNT/B cleavessynaptobrevin, a 116-amino-acid protein betweenGln-76 and Phe-77.Likewise, tetanus toxin (TeNT) is produced by theanaerobic bacillus Clostridium tetani and causes theparalytic tetanus syndrome by blocking neurotransmit-ter release at central synapses. As for BoNT/B, thelight chain of tetanus neurotoxin (TeNT–L chain) hasbeen shown to be endowed with a highly selective zincendopeptidase activity which is also directed towardsthe single Gln-76–Phe-77 bond of synaptobrevin.TeNT is always the cause of a number of deaths inspite of the existence of a vaccine. In the case of BoNT,there is presently no therapy and thus, the infectionremains a significant health problem. One potentialsolution to reduce BoNT/B toxicity is to inhibit itsproteolytic activity by designing selective and potentinhibitors of this protease. This requires the screeningof a great number of potential inhibitors, and thedevelopment of a sensitive and rapid in vitro assay.Moreover, although they belong to the group of zinc-metallopeptidases and the catalytic site of the recentlycrystallised BoNT/B (Hanson and Stevens, 2000) doesnot differ significantly from that of other zinc metal-lopeptidases, such as neutral endopeptidase (Roques etal., 1993), it seems that long sequences of synaptobre-vin are required for an efficient cleavage of synaptobre-vin by TeNT, as well as by BoNT/B (Hanson andStevens, 2000).